In the cystic fibrosis lung, P. aeruginosa resides in anaerobic environments present in the mucous airway plugs and in biofilms on the airway epithelium. This suggests that P. aeruginosa possesses genes/gene products required for growth in oxygen-limited conditions. The ability of this organism to survive under anaerobic conditions has not been thoroughly investigated. We hypothesize that P. aeruginosa possesses essential genes required for anaerobic growth, and that these genes are expressed in biofilms and by P. aeruginosa from the sputum of CF patients. Identification of genes required by P. aeruginosa under anaerobic conditions will provide a better understanding of how this organism causes chronic lung infections in CF patients. To begin to identify genes required for anaerobic growth of P. aeruginosa, we have used transposon mutagenesis to select for P. aeruginosa that have lost the ability to grow anaerobically. Nine different mutants have been obtained thus far. In Specific aim 1, we will identify the genes that have been disrupted by transposon insertion. Additionally, unmarked mutants in the identified genes will be generated and characterized. In Specific aim 2, these mutants will be evaluated for their ability to form biofilms compared to the wild-type strain, to begin to understand the role of the respective gene products in biofilm formation and development. In Specific aim 3, we will evaluate whether the genes identified are expressed in biofilms and in sputum taken from CF patients with P. aeruginosa lung infections. Also, gfp reporter fusions will be constructed to monitor levels and patterns of gene expression within the biofilm. The data obtained from these studies will expand our knowledge of the anaerobic hysiology of P. aeruginosa and provide new insight into strategies to treat chronic lung infections caused by P. aeruginosa.